Exosomes are small vesicles (40-100 nm) secreted by various cells and found in body fluids. They have been proposed as vehicles for microRNA (miRNA)-based intercellular communication and sources of miRNA biomarkers. However, a quantitative analysis of miRNA abundance and stoichiometry in exosomes is lacking. This study quantified the number of exosomes and miRNA molecules in replicate samples from five sources: plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells. The results showed that, on average, there was far less than one miRNA molecule per exosome, even for the most abundant miRNAs. This suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are unlikely to function as vehicles for miRNA-based communication. The study also found that only a small minority (median = 2.5%) of the extracellular miRNA content in plasma is associated with exosomes prepared by standard methods. These findings challenge the hypothesis that exosomes are major carriers of miRNAs and suggest that current models of exosome-mediated miRNA communication may need revision. The study used nanoparticle tracking analysis (NTA) to quantify exosome numbers and sizes, and real-time PCR to measure miRNA abundance. The results indicate that exosomes contain very few miRNAs, and that most exosomes do not carry significant numbers of miRNAs. This has implications for the use of exosomes as vehicles for miRNA-based communication and as sources of biomarkers. The study also highlights the need for further research to understand the relationship between different classes of RNA and extracellular vesicles.Exosomes are small vesicles (40-100 nm) secreted by various cells and found in body fluids. They have been proposed as vehicles for microRNA (miRNA)-based intercellular communication and sources of miRNA biomarkers. However, a quantitative analysis of miRNA abundance and stoichiometry in exosomes is lacking. This study quantified the number of exosomes and miRNA molecules in replicate samples from five sources: plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells. The results showed that, on average, there was far less than one miRNA molecule per exosome, even for the most abundant miRNAs. This suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are unlikely to function as vehicles for miRNA-based communication. The study also found that only a small minority (median = 2.5%) of the extracellular miRNA content in plasma is associated with exosomes prepared by standard methods. These findings challenge the hypothesis that exosomes are major carriers of miRNAs and suggest that current models of exosome-mediated miRNA communication may need revision. The study used nanoparticle tracking analysis (NTA) to quantify exosome numbers and sizes, and real-time PCR to measure miRNA abundance. The results indicate that exosomes contain very few miRNAs, and that most exosomes do not carry significant numbers of miRNAs. This has implications for the use of exosomes as vehicles for miRNA-based communication and as sources of biomarkers. The study also highlights the need for further research to understand the relationship between different classes of RNA and extracellular vesicles.